Project Summary Over the past half-century, biologists have generated a critical mass of knowledge and many compelling hypotheses about how chromatin, a collection of proteins and RNA scaffolded to DNA, regulates gene expression. This knowledge has the potential to transform our understanding of development and disease and spur new therapeutic strategies. To realize this potential, new experimental tools are needed to directly test existing hypotheses and reveal the functions of chromatin components. Of particular use would be approaches that directly and specifically track the biochemical and biophysical states of chromatin in live cells. This would unlock our ability to study dynamic changes in chromatin state in response to cellular perturbations. Observing intracellular events in temporal sequence would also enable functional studies demonstrating changes in gene regulation in direct response to changes in chromatin state. Towards this goal, we will develop small proteins that bind specific biochemical chromatin modifications. These binders will be genetically encoded, enabling their use in live cell microscopy. The development of these chromatin binders will support a new frontier in chromatin research. They will build upon the information garnered from static maps of chromatin state and expand our understanding of chromatin into both the temporal and spatial dimensions.